(see Note 19). Next, withdraw 10 μL of SBTI from Tube 1 and
mix it with the buffer in Tube 2 by pipetting up and down
several times. Next, mix 10 μL from Tube 2 with the contents
of Tube 3, etc., until Tube 15 is reached. Take 10 μL out of
Tube 15 and discard it. Tube 16 is the 0-μM control, and thus
is not part of the dilution series. Finally, add 10 μL of the
100-nM stock of labeled α-CT to each of the 16 tubes. Incubate in the dark at room temperature for 30 min.
2. Fill 16 of the preferred style of capillaries (we used Standard
capillaries, as there was no difference between Premium and
Standard) with the incubated samples. Each PCR tube should
have about 10 μL remaining after filling; store them in the dark
at room temperature. Place the capillaries into the capillary tray
in numbered order, and then insert the tray into the MST
instrument.
3. If using MO.Control, initialize a new experiment in the
“Expert Mode” module. Provide the α-CT concentration and
the desired LED level to the software (we used 50% in this
example; this was optimized in step 3 of Subheading 3.2). Set
the MST power to “Low.” Initiate a new dilution series and
provide the top concentration of SBTI as 20,000. Manually
edit the concentration for Capillary 16 to be 0.4. For the time
periods of the various experimental phases, see step 6 of Subheading 3.2. Start the experiment and allow it to complete. The
software will automatically conduct a post-run capillary scan
(see Note 13). Repeat this entire step two more times (with the
same capillaries in place) but using “Medium” and “High”
MST powers. Proceed to step 5.
4. If using NT.Control, perform a capillary scan using the LED
power optimized in Subheading 3.2 and insert the appropriate
concentrations for all capillaries (in nM units; Tube 1 is 20,000
in this example), except for Tube 16; divide the value in Tube
15 by approximately 3 and use this number (we inputted 0.4).
Remember to input the proper concentration of the labeled
α-CT, which is 50. Using the instrument parameters described
in step 6 of Subheading 3.2, collect three sequential data sets
using 20%, 40%, and 60% MST power. Collect a capillary scan
after each experiment (see Note 13).
5. Read the resulting data file into PALMIST, examining the 20%
MST-power experiment first. Press the “Cold Fluo.” button,
which presents a graph of the fluorescence before the IR laser
has been actuated (Fig. 4a). Is there a strong trend? If so, it is
possible that the fluorescence trend and not any thermophoretic phenomenon should be analyzed (see Note 20). In the
example case, there is a weak but noisy upward trend; this is
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Shih-Chia Tso and Chad A. Brautigam
mix it with the buffer in Tube 2 by pipetting up and down
several times. Next, mix 10 μL from Tube 2 with the contents
of Tube 3, etc., until Tube 15 is reached. Take 10 μL out of
Tube 15 and discard it. Tube 16 is the 0-μM control, and thus
is not part of the dilution series. Finally, add 10 μL of the
100-nM stock of labeled α-CT to each of the 16 tubes. Incubate in the dark at room temperature for 30 min.
2. Fill 16 of the preferred style of capillaries (we used Standard
capillaries, as there was no difference between Premium and
Standard) with the incubated samples. Each PCR tube should
have about 10 μL remaining after filling; store them in the dark
at room temperature. Place the capillaries into the capillary tray
in numbered order, and then insert the tray into the MST
instrument.
3. If using MO.Control, initialize a new experiment in the
“Expert Mode” module. Provide the α-CT concentration and
the desired LED level to the software (we used 50% in this
example; this was optimized in step 3 of Subheading 3.2). Set
the MST power to “Low.” Initiate a new dilution series and
provide the top concentration of SBTI as 20,000. Manually
edit the concentration for Capillary 16 to be 0.4. For the time
periods of the various experimental phases, see step 6 of Subheading 3.2. Start the experiment and allow it to complete. The
software will automatically conduct a post-run capillary scan
(see Note 13). Repeat this entire step two more times (with the
same capillaries in place) but using “Medium” and “High”
MST powers. Proceed to step 5.
4. If using NT.Control, perform a capillary scan using the LED
power optimized in Subheading 3.2 and insert the appropriate
concentrations for all capillaries (in nM units; Tube 1 is 20,000
in this example), except for Tube 16; divide the value in Tube
15 by approximately 3 and use this number (we inputted 0.4).
Remember to input the proper concentration of the labeled
α-CT, which is 50. Using the instrument parameters described
in step 6 of Subheading 3.2, collect three sequential data sets
using 20%, 40%, and 60% MST power. Collect a capillary scan
after each experiment (see Note 13).
5. Read the resulting data file into PALMIST, examining the 20%
MST-power experiment first. Press the “Cold Fluo.” button,
which presents a graph of the fluorescence before the IR laser
has been actuated (Fig. 4a). Is there a strong trend? If so, it is
possible that the fluorescence trend and not any thermophoretic phenomenon should be analyzed (see Note 20). In the
example case, there is a weak but noisy upward trend; this is
170
Shih-Chia Tso and Chad A. Brautigam